Lack of SPNS1 results in accumulation of lysolipids and lysosomal storage disease in mouse models
Hoa T.T. Ha, SiYi Liu, Xuan T.A. Nguyen, Linh K. Vo, Nancy C.P. Leong, Dat T. Nguyen, Shivaranjani Balamurugan, Pei Yen Lim, YaJun Wu, Eunju Seong, Toan Q. Nguyen, Jeongah Oh, Markus R. Wenk, Amaury Cazenave-Gassiot, Zuhal Yapici, Wei-Yi Ong, Margit Burmeister, Long N. Nguyen
Hoa T.T. Ha, SiYi Liu, Xuan T.A. Nguyen, Linh K. Vo, Nancy C.P. Leong, Dat T. Nguyen, Shivaranjani Balamurugan, Pei Yen Lim, YaJun Wu, Eunju Seong, Toan Q. Nguyen, Jeongah Oh, Markus R. Wenk, Amaury Cazenave-Gassiot, Zuhal Yapici, Wei-Yi Ong, Margit Burmeister, Long N. Nguyen
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Research Article Aging Metabolism

Lack of SPNS1 results in accumulation of lysolipids and lysosomal storage disease in mouse models

Abstract

Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1-KO cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global KO of Spns1 caused embryonic lethality between E12.5 and E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1-KO mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K/AKT signaling pathway. Furthermore, we identified 3 human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.

Authors

Hoa T.T. Ha, SiYi Liu, Xuan T.A. Nguyen, Linh K. Vo, Nancy C.P. Leong, Dat T. Nguyen, Shivaranjani Balamurugan, Pei Yen Lim, YaJun Wu, Eunju Seong, Toan Q. Nguyen, Jeongah Oh, Markus R. Wenk, Amaury Cazenave-Gassiot, Zuhal Yapici, Wei-Yi Ong, Margit Burmeister, Long N. Nguyen

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Figure 3

SPNS1 is required for sphingosine and LPC release from lysosomes.

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SPNS1 is required for sphingosine and LPC release from lysosomes. (A–C) ...
(A–C) Total levels of sphingosines from WT and Spns1-KO cells with or without starvation condition. WT and Spns1-KO CHO cells (A). WT and Spns1-KO HEK293 cells (B). WT, Spns1-KO, and NPC1-KO HEK293 cells (C). Each symbol represents 1 replicate (A and B, 2-tailed unpaired t test, n = 4; C, 1-way ANOVA, n = 4; *P < 0.05, ***P < 0.001, ****P < 0.0001; data are expressed as mean ± SD). (D) Illustration of [3-3H]-sphingosine transport assays. Cells were starved in medium without amino acids and serum for 1 hour, andthey were then added with radioactive sphingosine and incubated until collecting for radioactive S1P isolation from other sphingolipids (Sph, Cer, and SM) for quantification. S1P, Sphingosine-1-phosphate; Sph, Sphingosine; Cer, Ceramide; SM, Sphingomyelin. (E) Radioactive S1P levels (upper phase) from WT, Spns1-KO, and rescue CHO cells with mouse SPNS1 (mSPNS1) or human SPNS1 (hSPNS1). Each symbol represents 1 replicate. One-way ANOVA; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001; data are expressed as mean ± SD). (F) Illustration of NBD-palmitate labeling experiment. (G) Thin-layer chromatography (TLC) analysis of NBD-labeled phospholipids after 24 hours of pulse-labeling with NBD-palmitate in WT and Spns1-KO CHO cells. Arrowhead indicates NBD-LPC band. NL, neutral lipids. (H) Quantification of NBD-LPC from the TLC plate (2-tailed unpaired t test; n = 3; ***P < 0.001; data are expressed as mean ± SD). (I) LPC transport activity of mouse (mSPNS1), human SPNS1 (hSPNS1) in HEK293 cells, and the missense mutation P295L. E164K was used as a control. piRESv2-EGFP was used as a mock control. Transfected HEK293 cells were incubated with 10 μM [14C] LPC for 20 minutes and collected for quantification of radioactive signals. Each symbol represents 1 replicate (1-way ANOVA; n = 3; ****P < 0.001; data are expressed as mean ± SD).